Aurora A kinase inhibition compromises its antitumor efficacy by elevating PD-L1 expression

Aurora A plays a critical role in G2/M transition and mitosis, making it an attractive target for cancer treatment. Aurora A inhibitors showed remarkable antitumor effects in preclinical studies, but unsatisfactory outcomes in clinical trials have greatly limited their development. In this study, the Aurora A inhibitor alisertib upregulated programmed death ligand 1 (PD-L1) expression in a panel of tumor cells both in vitro and in vivo. Upregulation of the checkpoint protein PD-L1 reduced antitumor immunity in immune-competent mice, paradoxically inhibiting the antitumor effects of alisertib. Mechanistically, Aurora A directly bound to and phosphorylated cyclic GMP-AMP synthase (cGAS), suppressing PD-L1 expression in tumor cells. Aurora A inhibition by alisertib activated the cGAS/stimulator of IFN genes (STING)/NF-κB pathway and promoted PD-L1 expression. Combining alisertib with anti–PD-L1 antibody improved antitumor immunity and enhanced the antitumor effects of alisertib in immune-competent mice. Our results, which reveal the immunomodulatory functions of Aurora A inhibitors and provide a plausible explanation for the poor clinical outcomes with their use, offer a potential approach to improve the antitumor efficacy of these inhibitors.


High-throughput flow cytometry screening system
BxPC3 cells were seeded in a 96-well plate with a Multidrop Combi robotic dispenser (Thermo Fisher). After incubation at 37°C overnight, cells were treated with the compound and 500 IU/mL IFNγ (#TL-105, Beijing T&L Biotechnology) for 48 hours. Cells were then digested into single cells and incubated with APC-conjugated anti-human CD274 antibodies (#329708, Biolegend) at room temperature for 30 minutes. Samples were analyzed on the IntelliCyt iQue Screener PLUS (Sartorius).

Flow Cytometry
Cells were suspended in Stain Buffer (#554657, BD Pharmingen) and incubated with primary conjugated antibodies at room temperature for 30 minutes. After washing in Stain Buffer, samples were analyzed with a BD LSRFortessa cell analyzer (BD Bioscience, USA).

Western Blotting
Cells were seeded in six-well plates and treated as described in the figure legends with the addition of SDS loading buffer (6x) and boiled at 100°C for 10 minutes. Cell lysates were subjected to SDS-PAGE and transferred to a nitrocellulose membrane (#66485, Pall). The membrane was blocked with 5% non-fat milk at room temperature for 1 hour after protein transfer. After incubation with the primary antibody overnight, the membrane was washed with PBS-T and incubated with HRP-conjugated antibody in 5% non-fat milk for 1 hour at room temperature. Finally, ECL substrate was used for exposure. The images of full, uncut gels were shown in online Supplemental Material.

Recombinant human PD-1-Fc protein purification
Plasmid P8400-IL2-exPD-1-hFc was transfected into HEK293T cells using PEI transfection reagent, and the supernatant was collected after 72 hours. It was then lightly centrifuged, and debris of cells were filtered using a 0.

Immunohistochemistry
For immunohistochemistry, human colon tumor tissue microarrays were obtained from Zhongshan Hospital, Fudan University, and mouse tumor tissues were obtained from tumor xenografts. In brief, sections were deparaffinized, and antigens were restored in Sodium citrate antigen retrieval solution (pH 6.0). Endogenous peroxidases were blocked in 3% H2O2 solution. Tissue samples were incubated with specific primary antibodies and an HRP-labeled secondary antibody and then developed using DAB reagent (Servicebio, Wuhan). Nuclei were counterstained with Hematoxylin staining solution. The histological staining results were scored according to the staining intensity and classified into three grades: negative, low, and high.

RNA extraction and quantitative Real Time PCR
Total RNA extraction reagent (Vazyme) was used to isolate total RNA from cells. cDNA was obtained by reverse transcription using HiScript II Q RT SuperMix (Vazyme) according to the manufacturer's protocol. Gene expression was measured by quantitative RT-PCR on the Quant Studio 6 Flex Real-Time PCR system (ABI). Expression of target genes was normalized to that of GAPDH and calculated using the ∆∆Ct method. The primers for each target gene were shown in Table S6.

Knockdown and knockout
Viral material was packaged by transfecting packaging plasmids PAX2, PGMD2G, and sgRNA expression plasmids with a specific target to each gene to 293T cells. After 8 hours, the medium was replaced with DMEM containing 10% FBS. The supernatant was collected after transfecting for 48 hours and 72 hours. The two supernatants were then mixed, lightly centrifuged, and filtered using a 0.45-μm filter. The viruses were then added to the target cells with 1 μg/ml polybrene. After 48 hours, the medium was completely replaced with medium containing 1 μg/ml puromycin. After 72 hours of puromycin selection, the resistant cells were harvested as knockdown cells. For the knockout cells, puromycin-resistant cells were diluted for colony formation. After the single colony had grown to an appropriate size, genomic DNA was extracted. The region near the guide RNA was amplified by PCR, the PCR products were separated by agarose gel electrophoresis, and DNA fragments of the correct size were collected and sequenced.
The efficacy of knockdown and knockout were confirmed by western blotting.

Cytosolic DNA quantification
The concertrations of cytosolic DNA in cell lysate was detected by QuantiFluor dsDNA Sample Kit (E2671, Promega) according to the manufacturer's recommended procedure.